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成纤维细胞直接编程为心肌细胞 Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by D

Direct Reprogramming of Fibroblasts

into Functional Cardiomyocytes

by De?ned Factors

Masaki Ieda,1,2,3,6,*Ji-Dong Fu,1,2,3Paul Delgado-Olguin,1,2,4Vasanth Vedantham,1,5Yohei Hayashi,1

Benoit G.Bruneau,1,2,4and Deepak Srivastava1,2,3,*

1Gladstone Institute of Cardiovascular Disease

2Department of Pediatrics

3Department of Biochemistry and Biophysics

4Cardiovascular Research Institute

5Department of Medicine

University of California,San Francisco,San Francisco,CA94158,USA

6Present address:Departments of Cardiology and of Clinical and Molecular Cardiovascular Research,Keio University School of Medicine, Shinanomachi35,Shinjuku-ku,Tokyo160-8582,Japan

*Correspondence:ieda@cpnet.med.keio.ac.jp(M.I.),dsrivastava@https://www.wendangku.net/doc/4d13014017.html,(D.S.)

DOI10.1016/j.cell.2010.07.002

SUMMARY

The reprogramming of?broblasts to induced plurip-otent stem cells(iPSCs)raises the possibility that a somatic cell could be reprogrammed to an alterna-tive differentiated fate without?rst becoming a stem/ progenitor cell.A large pool of?broblasts exists in the postnatal heart,yet no single‘‘master regulator’’of direct cardiac reprogramming has been identi?ed. Here,we report that a combination of three develop-mental transcription factors(i.e.,Gata4,Mef2c,and Tbx5)rapidly and ef?ciently reprogrammed post-natal cardiac or dermal?broblasts directly into differ-entiated cardiomyocyte-like cells.Induced cardio-myocytes expressed cardiac-speci?c markers,had a global gene expression pro?le similar to cardio-myocytes,and contracted spontaneously.Fibro-blasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells.We believe these?nd-ings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by de?ned factors.Reprogramming of endogenous or explanted?broblasts might pro-vide a source of cardiomyocytes for regenerative approaches.

INTRODUCTION

Heart disease is a leading cause of adult and childhood mortality. The underlying pathology is typically loss of cardiomyocytes that leads to heart failure or improper development of cardiomyo-cytes during embryogenesis that leads to congenital heart malformations.Because postnatal cardiomyocytes have little or no regenerative capacity,current therapeutic approaches are limited.Embryonic stem cells possess clear cardiogenic potential,but ef?ciency of cardiac differentiation,risk of tumor formation,and issues of cellular rejection must be overcome (Ivey and Srivastava,2006;La?amme et al.,2007;Nussbaum et al.,2007;van Laake et al.,2008).The ability to reprogram ?broblasts into induced pluripotent stem cells(iPSCs)with four de?ned factors might address some of these issues by providing an alternative source of embryonic-like stem cells(Takahashi and Yamanaka,2006).However,generating suf?cient iPSC-derived cardiomyocytes that are pure and mature and that can be delivered safely remains challenging(Zhang et al.,2009). The human heart is composed of cardiomyocytes,vascular cells,and cardiac?broblasts.In fact,cardiac?broblasts com-prise over50%of all the cells in the heart(Baudino et al., 2006;Camelliti et al.,2005;Snider et al.,2009).Cardiac?bro-blasts are fully differentiated somatic cells that provide support structure,secrete signals,and contribute to scar formation upon cardiac damage(Ieda et al.,2009).Fibroblasts arise from an extracardiac source of cells known as the proepicardium, and do not normally have cardiogenic potential(Snider et al., 2009).The large population of endogenous cardiac?broblasts is a potential source of cardiomyocytes for regenerative therapy if it were possible to directly reprogram the resident?broblasts into beating cardiomyocytes.Unfortunately,although embryonic mesoderm can be induced to differentiate into cardiomyocytes (Takeuchi and Bruneau,2009),efforts to accomplish this in somatic cells have thus far been unsuccessful,and to our knowl-edge,no‘‘master regulator’’of cardiac differentiation,like MyoD for skeletal muscle(Davis et al.,1987),has been identi?ed to date.

The generation of iPSCs suggests that a speci?c combination of de?ned factors,rather than a single factor,could epigeneti-cally alter the global gene expression of a cell and allow greater plasticity of cell type than previously appreciated.Consistent Cell142,375–386,August6,2010a2010Elsevier Inc.375

with this,the bHLH transcription factor,Neurogenin 3,in combi-nation with Pdx1and Mafa,can ef?ciently reprogram pancreatic exocrine cells into functional b cells in vivo,although the exocrine cells were known to have some potential to become islet cells in vitro and share a common parent cell with islet cells (Baeyens et al.,2005;Zhou et al.,2008).A combination of three factors,Ascl1,Brn2,and Myt1l,converts dermal ?broblasts to functional neurons (Vierbuchen et al.,2010),although the degree of global reprogramming of the neurons is unknown.

In this study,we examined whether key developmental cardiac regulators could reprogram cardiac ?broblasts into car-diomyocytes.We found that out of a total of 14factors,a speci?c combination of three transcription factors,Gata4,Mef2c,and Tbx5,was suf?cient to generate functional beating cardiomyo-cytes directly from mouse postnatal cardiac or dermal ?bro-blasts and that the induced cardiomyocytes (iCMs)were globally reprogrammed to adopt a cardiomyocyte-like gene expression pro?le.

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Figure 1.Screening for Cardiomyocyte-Inducing Factors

(A)Schematic representation of the strategy to test

candidate cardiomyocyte-inducing factors.

(B)Morphology and characterization of ?broblast-like cells migrating from a MHC-GFP heart explants.Phase contrast (left),GFP (middle),and Thy-1immunostaining (right).Insets are high-magni?cation views.See also Figure S1.

(C)Thy-1+/GFP àcells were FACS sorted from explant cultures for reprogramming.

(D)Summary of FACS analyses for a -MHC-GFP +cells.Effect on GFP +cell induction with 14factors or the removal of individual factors from the pool of 14factors (n =3).Removal of Baf60c,Hand2,Hopx,Hrt2,or Pitx2c did not decrease the percent of GFP +cells and were excluded for further anal-yses.See also Figure S2.

(E)FACS plots for analyses of GFP +cells.GFP +cells were analyzed 1week after 14factor trans-duction.The number of GFP +cells were reduced by removal of Gata4,but increased by removal of Pitx2c from 14factors.

(F–H)Effect on GFP +cell induction of the removal of individual factors from the pool of 9(F),6(G),or 5(H)factors (n =3).Factors that did not decrease ef?ciency upon removal were excluded from further study.

(I)GFP +(20%)cells were induced from ?broblasts by the combination of four factors,Gata4,Mef2c,Mesp1,and Tbx5.Representative data are shown in each panel.PI,propidium iodine.All data are pre-sented as means ±SD.*p <0.01;**p <0.05versus relevant control.Scale bars represent 100m m.See also Figures S1and S2.

RESULTS

Screening for Cardiomyocyte-Inducing Factors

We developed an assay system in which the induction of mature cardiomyo-cytes from ?broblasts could be analyzed quantitatively by reporter-based ?uorescence-activated cell sorting (FACS)(Fig-ure 1A).To accomplish this,we generated a MHC promoter-driven EGFP-IRES-puromycin transgenic mice (a MHC-GFP),in which only mature cardiomyocytes expressed the green ?uores-cent protein (GFP)(Gulick et al.,1991).We con?rmed that only cardiomyocytes,but not other cell types such as cardiac ?bro-blasts,expressed GFP in the transgenic mouse hearts and in primary cultured neonatal mouse cardiac cells (Figure S1avail-able online).

To have enough cardiac ?broblasts for FACS screening,we obtained GFP àcardiac ?broblasts from neonatal a MHC-GFP hearts by explant culture.Fibroblast-like cells migrated from the explants after 2days and were con?uent after 1week.The migrating cells did not express GFP,but expressed Thy1and vimentin,markers of cardiac ?broblasts (Figure 1B and data not shown)(Hudon-David et al.,2007;Ieda et al.,2009).To avoid contamination of cardiomyocytes,we ?ltered the cells by cell

376Cell 142,375–386,August 6,2010a2010Elsevier Inc.

strainers to remove heart tissue fragments and isolated Thy1+/ GFPàcells by FACS(Figure1C).Using FACS,we con?rmed that Thy1+/GFPàcells did not express cardiac troponin T(cTnT), a speci?c sarcomeric marker of differentiated mature cardio-myocytes(Figure S1)(Kattman et al.,2006).With these proce-dures,we had no cardiomyocyte contamination in the?broblast culture and could generate greater than twice the number of cardiac?broblasts than by conventional?broblast isolation tech-niques(Ieda et al.,2009).

To select potential cardiac reprogramming factors,we used microarray analyses to identify transcription factors and epige-netic remodeling factors with greater expression in mouse cardiomyocytes than in cardiac?broblasts at embryonic day 12.5(Ieda et al.,2009).Among them,we selected13factors that exhibited severe developmental cardiac defects and embryonic lethality when mutated(Figure S2).We also included the cardiovascular mesoderm-speci?c transcription factor Mesp1because of its cardiac transdifferentiation effect in Xenopus(David et al.,2008).We generated individual retrovi-ruses to ef?ciently express each gene in cardiac?broblasts (Figure S2).

We transduced Thy1+/GFPàneonatal mouse cardiac?bro-blasts with a mixture of retroviruses expressing all14factors or with DsRed retrovirus(negative control)(Hong et al.,2009). We did not observe any GFP+cells in cardiac?broblasts1week after Ds-Red retrovirus infection or1week of culture without any viral infection.In contrast,transduction of all14factors into?bro-blasts resulted in the generation of a small number of GFP+cells (1.7%),indicating the successful activation of the cardiac-enriched a MHC gene in some cells(Figures1D and1E).

To determine which of the14factors were critical for activating cardiac gene expression,we serially removed individual factors from the pool of14.Pools lacking?ve factors(Baf60c,Hand2, Hopx,Hrt2,and Pitx2c)produced an increased number of GFP+cells,suggesting they are dispensable in this setting (Figures1D and1E).Of note,removing Gata4decreased the percentage of GFP+cells to0.5%,and removing Pitx2c increased it to5%.Removal of the?ve factors listed above resulted in an increase in the percentage of GFP+cells to13% (Figure1F).We conducted three further rounds of withdrawing single factors from nine-,six-,and?ve-factor pools,removing those that did not decrease ef?ciency upon withdrawal,and found that four factors(Gata4,Mef2c,Mesp1,and Tbx5)were suf?cient for ef?cient GFP+cell induction from cardiac?bro-blasts(Figures1F–1H).The combination of these four factors dramatically increased the number of?broblasts activating the a MHC-GFP reporter to over20%(Figure1I).

Gata4,Mef2c,and Tbx5Are Suf?cient for Cardiomyocyte Induction

Next,we examined the expression of cTnT by FACS.We found that20%of GFP+cells expressed cTnT at high enough levels to detect by FACS1week after the four-factor transduction. Again removing individual factors from the four-factor pool in transduction,we found that Mesp1was dispensable for cTnT expression(Figures2A and2B).In contrast,we did not observe cTnT+or GFP+cells,when either Mef2c or Tbx5was removed. Removal of Gata4did not signi?cantly affect the number of GFP+cells,but cTnT expression was abolished,suggesting Gata4was also required.Whereas the combination of two factors,Mef2c and Tbx5,induced GFP expression but not cTnT,no combination of two factors or single factor induced both GFP and cTnT expression in cardiac?broblasts(Figure2C). These data suggested that the combination of three factors, Gata4,Mef2c,and Tbx5,is suf?cient to induce cardiac gene expression in?broblasts.

We found that30%of GFP+cells expressed cTnT1week after the three-factor transduction.Next,to con?rm our screening results,we transduced cardiac?broblasts with three factors (Gata4,Mef2c,and Tbx5,hereafter referred to as GMT)plus Nkx2-5,a critical factor for cardiogenesis but excluded by our initial screening.Surprisingly,adding Nkx2-5to GMT dramati-cally inhibited the expression of GFP and cTnT in cardiac ?broblasts.We also transduced cardiac?broblasts with the combination of Baf60c,Gata4,and Tbx5,which can transdiffer-entiate noncardiogenic mesoderm to cardiomyocytes in mouse embryos(Takeuchi and Bruneau,2009).We found that this combination did not ef?ciently induce cTnT or GFP expression above that of Tbx5alone,con?rming our screening results (Figure2D).

To determine if other cardiac genes were enriched in GFP+ cells,we sorted GFP+cells and GFPàcells7days after transduc-tion with GMT and compared gene expression of cardiomyo-cyte-speci?c genes,Myh6(a-myosin heavy chain),Actc1 (cardiac a-actin),Actn2(actinin a2),and Nppa(natriuretic peptide precursor type A)by quantitative RT-PCR(qPCR).We found that these cardiac genes were upregulated signi?cantly more in GFP+than in GFPàcells(Figure2E).Next,we used immunocytochemistry to determine if cardiac proteins were expressed in GFP+cells.Despite the detection of cTnT in only 30%of GFP+cells,most GFP+cells induced with the three fac-tors expressed sarcomeric a-actinin(a-actinin)and had well-de?ned sarcomeric structures,similar to neonatal cardiomyo-cytes(Figure2F;Figure S1).In addition to a-actinin,some GFP+cells also expressed cTnT and ANF(atrial natriuretic factor),indicating GFP+cells expressed several cardiomyo-cyte-speci?c markers(Figures2G and2H).We also con?rmed that neither GFP+nor GFPàcells expressed smooth muscle or endothelial cell markers(Figure S2),suggesting speci?city of GMT effects.

Induced Cardiomyocytes Originate from Differentiated Fibroblasts and Are Directly Reprogrammed

We next isolated neonatal cardiac?broblasts by the conven-tional?broblast isolation method in which hearts were digested with trypsin and plated on plastic dishes(Ieda et al.,2009). More than85%of the cells expressed Thy1,and we isolated Thy1+/GFPàcells by FACS to exclude cardiomyocyte contami-nation(Figure3A).Fibroblasts transduced with GMT expressed GFP,cTnT,and actinin after1week at the same level as?bro-blasts isolated from explant cultures(Figures3B and3C).Similar results were obtained on introduction of GMT into adult cardiac ?broblasts,with full formation of sarcomeric structures(Fig-ure3D;Figure S2).

To determine if the induced cardiomyocyte-like cells(iCMs) were arising from a subpopulation of stem-like cells,we Cell142,375–386,August6,2010a2010Elsevier Inc.377

analyzed c-kit expression (Beltrami et al.,2003)in the Thy1+/GFP àcells.Most c-kit +cells coexpressed Thy1,whereas 15%of Thy1+cells expressed c-kit,which is consistent with a previous report of cardiac explant-derived cells (Davis et al.,2009).We isolated GFP à/Thy1+/c-kit +cells and GFP à/Thy1+/c-kit àcells by FACS and transduced each population of cells with GMT.We found 2–3-fold more cardiomyocyte induction in GFP à/Thy1+/c-kit àcells than in GFP à/Thy1+/c-kit +cells (Figure S3).These results suggest that most of the iCMs originated from a c-kit-negative population.

We then sought to more de?nitively exclude the possibility of rare cardiac progenitors giving rise to iCMs.We tested the potential of mouse tail-tip dermal ?broblasts to generate iCMs.We found that sorted Thy1+/GFP àtail-tip dermal ?broblasts transduced with GMT expressed GFP at the same level as GMT-transduced cardiac ?broblasts,although the percentage of cTnT +cells was less than cardiac ?broblast-derived iCMs

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Figure https://www.wendangku.net/doc/4d13014017.html,bination of Three Transcrip-tion Factors Induces Cardiac Gene Expres-sion in Fibroblasts

(A)FACS analyses for a -MHC-GFP and cardiac Troponin T (cTnT)expression.Effects of the removal of individual factors from the pool of four factors on GFP +and cTnT +cell induction.

(B)Quantitative data of GFP +cells and cTnT +cells in (A)(n =3).

(C)Effect of the transduction of pools of three,two,and one factors on GFP +and cTnT +cell induction (n =3).

(D)FACS analyses for a -MHC-GFP and cTnT expression.Effects of GMT plus Nkx2.5and Baf60c/Gata4/Tbx5transduction are shown.

(E)The mRNA expression in GFP +and GFP àcells 7days after GMT transduction was determined by qPCR (n =3).

(F)Immuno?uorescent staining for GFP,a -actinin,and DAPI.The combination of the three factors,GMT,induced abundant GFP,and a -actinin expression in cardiac ?broblasts 2weeks after transduction.High-magni?cation views in insets show sarcomeric organization.See also Figures S1and S2.

(G)Induced cardiomyocytes expressed cTnT by immunocytochemistry with clear sarcomeric orga-nization 4weeks after transduction.Insets are high-magni?cation views.

(H)Induced cardiomyocytes expressed ANF at perinuclear sites 2weeks after transduction.All data are presented as means ±SD.*p <0.01versus relevant control.Scale bars represent 100m m.

See also Tables S1and S2and Figure S4.

We next compared the progressive global gene expression pattern of iCMs,neonatal cardiomyocytes,and cardiac ?broblasts by mRNA microarray anal-yses.We sorted GFP +cells and GFP àcells 2and 4weeks after GMT transduc-tion.The iCMs at both stages were similar to neonatal cardiomyocytes,but were distinct from GFP à

cells and cardiac ?broblasts in global gene expression pattern (Figure 4E).We found that functionally impor-tant cardiac genes were upregulated signi?cantly more in 4week iCMs than in 2week iCMs,including Pln (phospholamban),Slc8a1(sodium/calcium exchanger),Myh6,Sema3a (sema-phorin 3a),Id2(inhibitor of DNA binding 2),and Myl2(myosin,light polypeptide 2,regulatory,cardiac,slow,also known as MLC2v)(Table S1).Conversely,some genes were downregulated more in 4week iCMs than in 2week iCMs (Table S1).The array analyses also identi?ed genes that were upregulated more in neonatal car-diomyocytes than in 4week iCMs or cardiac ?broblasts (group 1in Figure 4E),including Bmp10(bone morphogenetic protein 10),Erbb4(v-erb-a erythroblastic leukemia viral oncogene homolog 4),Irx4(Iroquois related homeobox 4),and Atp1a2(ATPase,Na +/K +transporting,a 2polypeptide)(Table S2).We also identi-?ed genes that were expressed to a greater extent in both cardi-omyocytes and 4week iCMs than in ?broblasts (group 2in

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Figure 4E),including Actc1,Myl7(myosin,light polypeptide 7,regulatory,also known as MLC2a),Tnnt2(troponin T2,cardiac),Tbx3(T-box 3),and Srf (serum response factor)(Table S2).Thus,iCMs were similar,but not identical,to neonatal cardiomyo-cytes,and the reprogramming event was broadly re?ected in global gene expression changes.

Fibroblasts Are Epigenetically Reprogrammed

to a Cardiomyocyte-like State by Gata4/Mef2c/Tbx5

To determine if iCMs have gained a cardiomyocyte-like chro-matin state,we analyzed the enrichment of histone modi?cations in the promoter regions of the cardiac-speci?c genes Actn2,Ryr2I ,and Tnnt2.We analyzed the enrichment of trimethylated histone H3of lysine 27(H3K27me3)and lysine 4(H3K4me3),which mark transcriptionally inactive or active chromatin,respectively (Li et al.,2007),in cardiac ?broblasts,4week iCMs,and neonatal cardiac cells by chromatin immunoprecipita-tion,followed by qPCR (Figure 5A).After reprogramming,H3K27me3was signi?cantly depleted at the promoters of all the genes analyzed in iCMs,reaching levels comparable to those in cardiac cells,whereas H3K4me3increased on the promoter regions of Actn2and Tnnt2in iCMs,as compared with cardiac ?broblasts.Ryr2had similar levels of H3K4me3in iCMs as in ?broblasts,suggesting that its activation re?ects the resolution of a ‘‘bivalent’’chromatin mark (Bernstein et al.,2006).These results suggested that cardiac ?broblast-derived iCMs gained a chromatin status similar to cardiomyocytes at least in some cardiac speci?c genes.Intriguingly,H3K27me3levels were higher in tail-tip ?broblasts than cardiac ?broblasts on all three genes analyzed and,despite a signi?cant reduction upon reprogramming to iCMs,remained somewhat higher than in cardiac cells and cardiac ?broblast-derived iCMs.

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Figure 5.Fibroblasts Are Stably Reprog-rammed into iCMs by Gata4,Mef2c,and Tbx5

(A)The promoters of Actn2,Ryr2,and Tnnt2were analyzed by ChIP for trimethylation status of histone H3of lysine 27or 4in cardiac ?broblasts (CF),CF-derived iCMs,tail-tip ?broblasts (TTF),TTF-derived iCMs,and neonatal cardiac cells.Data were quanti?ed by qPCR.

(B)The promoters of Nppa and Myh6were analyzed with bisul?te genomic sequencing for DNA methylation status in CF,a -MHC-GFP àcells,a -MHC-GFP +iCMs (FACS sorted 4weeks after transduction),and neonatal CM.Open circles indi-cate unmethylated CpG dinucleotides;closed circles indicate methylated CpGs.

(C)Schematic representation of the strategy to test expression kinetics of the doxycycline (Dox)-inducible lentiviral system.

(D)Wild-type TTFs were infected with pLVX-tetO-GFP and pLVX-rtTA and imaged before (off Dox),1day after Dox addition,and at time points after Dox withdrawal (–Dox).All images were taken using constant exposure times and identical camera settings.

(E)Schematic representation of the strategy to determine temporal requirement of Gata4/Mef2c/Tbx5in reprogramming.Thy1+/GFP àTTF were infected with the pLVX-tetO-GMT and pLVX-rtTA lentiviruses,and Dox was added for 2weeks and thereafter withdrawn for 1week.

(F)Immuno?uorescent staining for GFP,a -actinin,and DAPI in iCMs 2weeks after lentiviral infection and Dox induction.

(G)Immuno?uorescent staining for GFP,a -actinin,and DAPI 1week after Dox withdrawal.iCMs maintained a -MHC GFP expression and had a -actinin positive sarcomeric structures.High-magni?cation views in insets show sarcomeric organization.Representative data are shown in each panel.All data are presented as means ±SD.*p <0.01;**p <0.05versus relevant control.Scale bars represent 100m m.

Cell 142,375–386,August 6,2010a2010Elsevier Inc.381

The DNA methylation status of speci?c loci also re?ects the stability of the reprogramming event and we therefore investi-gated such changes during reprogramming from cardiac?bro-blasts to iCMs.We performed bisul?te genomic sequencing in the promoter regions of Nppa and Myh6in cardiac?broblasts, 4week GFPàcells,iCMs,and neonatal cardiomyocytes.Both promoter regions were hypermethylated in cardiac?broblasts and GFPàcells,as expected from the cardiomyocyte-speci?c expression of these genes,but were comparatively demethy-lated in iCMs,similar to neonatal cardiomyocytes(Figure5B). These results indicated that reprogramming by Gata4,Mef2c, and Tbx5induced epigenetic resetting of the?broblast genome to a cardiomyocyte-like state.

To further assess the stability of the reprogramming event,we generated a doxycycline-inducible lentiviral system in which transgene expression of the reprogramming factors was con-trolled by doxycycline administration.We?rst transduced wild-type tail-tip?broblasts with a mixture of lentiviruses containing pLVX-tetO-GFP and pLVX-rtTA to determine the expression kinetics of this system(Figure5C).We con?rmed that the majority of?broblasts infected with both viruses expressed GFP within1day after doxycycline induction,and the GFP expression was instantly diminished by withdrawal of doxycy-cline and disappeared within6days(Figure5D).Thy1+/GFPàtail-tip?broblasts were harvested from a MHC-GFP neonatal mice,transduced with a pool of lentiviruses containing inducible Gata4,Mef2c,and Tbx5,along with pLVX-rtTA,and subse-quently treated with doxycycline(Figure5E).We found that a MHC-GFP expression was induced from tail-tip?broblasts after doxycycline administration and that the iCMs had well-de?ned sarcomeric structures marked with an anti-a-actinin antibody after2weeks of culture(Figure5F).Doxycycline was withdrawn after2weeks of culture,and cells were subsequently cultured without doxycycline for1week to fully remove exoge-nous expression of the reprogramming factors(Figure5E).The iCMs maintained a MHC-GFP expression and had sarcomeric structures after doxycycline withdrawal,suggesting that the ?broblasts were stably reprogrammed into iCMs after2weeks exposure to Gata4,Mef2c,and Tbx5(Figure5G).

Induced Cardiomyocytes Exhibit

Spontaneous Contraction

To determine if iCMs possessed the functional properties char-acteristic of cardiomyocytes,we analyzed intracellular Ca2+?ux in iCMs after2–4weeks of culture.Around30%of cardiac ?broblast-derived iCMs showed spontaneous Ca2+oscillations and their frequency was variable,resembling what was observed in neonatal cardiomyocytes(Figures6A,6B,and6D;Movie S1). We observed that tail-tip dermal?broblast-derived iCMs also exhibited spontaneous Ca2+oscillations,but the oscillation frequency was lower than that of cardiomyocytes and cardiac ?broblast-derived iCMs(Figures6C and6E;Movie S2).

In addition to the characteristic Ca2+?ux,cardiac?broblast-derived iCMs showed spontaneous contractile activity after 4–5weeks in culture(Movies S3and S4;Figure S5).Single-cell extracellular recording of electrical activity in beating cells revealed tracings similar to the potential observed in neonatal cardiomyocytes(Figure6F).Intracellular electrical recording of iCMs displayed action potentials that resembled those of adult mouse ventricular cardiomyocytes(Figure6G).Thus,the reprog-ramming of?broblasts to iCMs was associated with global changes in gene expression,epigenetic reprogramming,and the functional properties characteristic of cardiomyocytes. Transplanted Cardiac Fibroblasts Transduced

with Gata4/Mef2c/Tbx5Reprogram In Vivo

To investigate whether GMT-transduced cardiac?broblasts can be reprogrammed to express cardiomyocyte-speci?c genes in their native environment in vivo,we harvested GFPà/Thy1+ cardiac?broblasts1day after viral transduction and injected them into immunosuppressed NOD-SCID mouse hearts.GMT-infected cells did not express GFP at the time of transplantation (Figure4A).Cardiac?broblasts were infected with either the mixture of GMT and DsRed retroviruses or DsRed retrovirus (negative control)to be readily identi?ed by?uorescence. Cardiac?broblasts infected with DsRed did not express a-acti-nin or GFP,con?rming cardiomyocyte conversion did not happen in the negative control(Figures7A and7B).Despite being injected into the heart only1day after viral infection, a subset of cardiac?broblasts transduced with GMT and DsRed expressed GFP in the mouse heart within2weeks(Figure7B). Importantly,the GFP+cells expressed a-actinin and had sarco-meric structures(Figure7C).These results suggested that cardiac?broblasts transduced with Gata4,Mef2c,and Tbx5 can reprogram to cardiomyocytes within2weeks upon trans-plantation in vivo.

DISCUSSION

Here we demonstrated that the combination of three transcrip-tion factors,Gata4,Mef2c,and Tbx5,can rapidly and ef?ciently induce cardiomyocyte-like cells from postnatal cardiac and dermal?broblasts.iCMs were similar to neonatal cardiomyo-cytes in global gene expression pro?le,electrophysiologically, and could contract spontaneously,demonstrating that func-tional cardiomyocytes can be generated from differentiated somatic cells by de?ned factors.Although much re?nement and characterization of the reprogramming process will be necessary,the?ndings reported here raise the possibility of reprogramming the vast pool of endogenous?broblasts that normally exists in the heart into functional cardiomyocytes for regenerative purposes.

The three reprogramming factors,Gata4,Mef2c,and Tbx5, are core transcription factors during early heart development (Olson,2006;Srivastava,2006;Zhao et al.,2008).They interact with one another,coactivate cardiac gene expression(e.g., Nppa,Gja5[Cx40],and Myh6),and promote cardiomyocyte differentiation(Bruneau et al.,2001;Garg et al.,2003;Ghosh et al.,2009;Lin et al.,1997).Gata4is considered a‘‘pioneer’’factor and might open chromatin structure in cardiac loci(Cirillo et al.,2002),thus allowing binding of Mef2c and Tbx5to their speci?c target sites and leading to full activation of the cardiac program.Although the reprogramming event appears stable at the epigenetic level,as marked by histone methylation and DNA methylation,the global gene expression of iCMs is similar but not identical to neonatal cardiomyocytes.Whether they are

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more similar to adult ventricular cardiomyocytes or other subpopulations remains to be determined.Additional epigenetic regulators,microRNAs,or signaling proteins may be leveraged

to increase the ef?ciency and robustness of the reprogramming event.Furthermore,other combinations of factors likely also induce cardiac reprogramming,much like the experience in the iPSC ?eld.

Several lines of evidence suggest that the iCMs we describe here originated from differentiated ?broblasts.We found that any potential rare cardiac ‘‘progenitor-like’’cells,marked by c-kit or Isl1,were dispensable for cardiomyocyte induction (Beltrami et al.,2003).Furthermore,the high ef?ciency of cardiac induction (up to 20%)does not favor the interpretation

that rare stem or progenitor cells were the origin of induced cardiomyocytes.Most importantly,the ability to reprogram dermal ?broblasts into iCMs supports the conclusion that cardiac progenitors are not the target cells for the reprogram-A

Ca 2+ max Ca 2+ min C

Rhod-3 intensity

(Tail tip fibroblast-derived iCMs)

Tail tip fibroblast-derived iCMs

Cardiac fibroblast-derived iCMs F Rhod-3 intensity (Neonatal cardiomyocytes)

1 s 1 s

Rhod-3 intensity (Cardiac fibroblast-derived iCMs)

1 s 1 s G

Cardiac fibroblast-derived iCMs

extracellular electrical recording

0 mV

Cardiac fibroblast-derived iCMs

50 ms

20 m V

1 s

Neonatal cardiomyocytes

1 s

2 m V

Neonatal cardiomyocytes extracellular electrical recording 1 s

2 m V

Figure 6.Induced Cardiomyocytes Exhibit Spontaneous Ca 2+Flux,Electrical Activity,and Beating

(A and B)Cardiac ?broblast (CF)-derived iCMs showed spontaneous Ca 2+oscillation with varying frequency (A),similar to neonatal cardiomyocytes (B).Rhod-3intensity traces are shown.

(C)Tail-tip dermal ?broblast (TTF)-derived iCMs showed spontaneous Ca 2+oscillation with lower frequency.The Rhod-3intensity trace is shown.(D)Spontaneous Ca 2+waves observed in CF-derived a -MHC-GFP +iCMs (white dots)or neonatal cardiomyocytes (arrows)with Rhod-3at Ca 2+max and min is shown.Fluorescent images correspond to the Movie S1.

(E)Spontaneous Ca 2+oscillation observed in the TTF-derived a -MHC-GFP +iCMs with Rhod-3at Ca 2+max and min is shown.Fluorescent images correspond to the Movie S2.

(F)Spontaneously contracting iCMs had electrical activity measured by single cell extracellular elec-trodes.Neonatal cardiomyocytes showed similar electrical activity.

(G)Intracellular electrical recording of CF-derived iCMs cultured for 10weeks displayed action potentials that resembled those of adult mouse ventricular cardiomyocytes.Representative data are shown in each panel (n =10in A–F,n =4in G).See also Figure S5and Movies S1,S2,S3and S4.

See also Movies S1,S2,S3,and S4and Figure S5.

ming factors.Remarkably,reprogram-ming of cardiac ?broblasts to myocytes occurred in a relatively short period,

with the ?rst GFP +cells appearing at day 3,in contrast to iPSC reprogram-ming,which typically takes 10–20days

and occurs with much lower ef?ciency (<0.1%)(Takahashi and Yamanaka,2006).Despite the early initiation of reprogramming,the process appears to continue for several weeks,with progres-sive changes in gene expression,contractile ability,and electro-physiologic maturation.

Although many questions remain regarding the mechanisms of reprogramming,we were able to genetically test the ‘‘route’’of cell fate alteration.Our ?ndings suggest that cardiomyocytes were directly induced from cardiac ?broblasts without reverting

to a cardiac progenitor cell state,which may explain the rapid early reprogramming process.This conclusion was supported by the absence of Isl1-Cre-YFP or Mesp1-Cre-YFP activation during the process of reprogramming,which would have marked any cells that transiently expressed Isl1or Mesp1(Laugwitz et al.,2005;Saga et al.,1999).

The ability to reprogram endogenous cardiac ?broblasts into cardiomyocytes has many therapeutic implications.First,the avoidance of reprogramming to pluripotent cells before cardiac differentiation would greatly lower the risk of tumor formation in the setting of future cell-based therapies.Second,large

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amounts of an individual’s own ?broblasts can be grown from a cardiac biopsy or skin biopsy in vitro for transduction with the de?ned factors,followed by delivery of cells to damaged hearts.Third,and most promising,is the potential to introduce the de?ned factors,or factors that mimic their effects,directly into the heart to reprogram the endogenous ?broblast popula-tion,which represents more than 50%of the cells,into new car-diomyocytes that can contribute to the overall contractility of the heart.Our observation that injection of ?broblasts into the heart only 1day after induction of Gata4/Mef2c/Tbx5resulted in reprogramming of the transplanted cells suggests that this may be possible.Future studies in human cells and advances in safe delivery of de?ned factors will be necessary to advance this technology for potential regenerative therapies.

EXPERIMENTAL PROCEDURES

Generation of a MHC-GFP,Isl1-YFP,and Mesp1-YFP Mice

To generate a MHC-GFP mice,EGFP-IRES-Puromycin cDNA was subcloned into the expression vector containing a -myosin heavy chain promoter (Gulick et al.,1991).Pronuclear microinjection and other procedures were performed according to the standard protocols (Ieda et al.,2007).PCR primers are listed in the Extended Experimental Procedures .Isl1-YFP mice were obtained by crossing Isl1-Cre mice and R26R-EYFP mice (Srinivas et al.,2001).Mesp1-YFP mice were obtained by crossing Mesp1-Cre mice and R26R-EYFP mice (Saga et al.,1999).

DsRed αMHC-GFP Merged

DsRed αMHC-GFP

Merged

αMHC-GFP Merged

GMT-cell injected

α-actinin

Merged

DsRed-cell injected DsRed-cell injected DsRed-cell injected

DsRed

α-actinin Figure 7.Transplanted Cardiac Fibroblasts Trans-duced with Gata4/Mef2c/Tbx5Can Be Reprog-rammed to Cardiomyocytes In Vivo

(A)DsRed infected cardiac ?broblasts (DsRed-cell)were transplanted into NOD-SCID mouse hearts 1day after infection and cardiac sections were analyzed by immuno-cytochemistry after 2weeks.Transplanted ?broblasts marked with DsRed did not express a -actinin (green).(B)Cardiac ?broblasts infected with DsRed or Gata4/Mef2c/Tbx5with DsRed (GMT/DsRed-cell)were trans-planted into NOD-SCID mouse hearts 1day after infection and visualized by histologic section.Note that a subset of GMT/DsRed cells expressed a -MHC-GFP.Data were analyzed 2weeks after transplantation.

(C)Gata4/Mef2c/Tbx5-transduced cardiac ?broblasts (GMT-cell)were transplanted into mouse hearts and histo-logic sections analyzed.A subset of induced GFP +cells expressed a -actinin (red)and had sarcomeric structures.Insets are high-magni?cation views of cells indicated by arrows.Data were analyzed 2weeks after transplantation.Representative data are shown in each panel (n =4in each group).Scale bars represent 100m m.Note that GMT/DsRed or GMT-infected cells did not express GFP at the time of transplantation (Figure 4A).

Cell Culture

For explant culture,isolated neonatal or adult mouse hearts were minced into small pieces less than 1mm 3in size.The explants were plated on gelatin-coated dishes and cultured for 7days in explant medium (IMDM/20%FBS)(Andersen et al.,2009).Migrated cells were har-vested and ?ltered with 40m m cell strainers (BD)to avoid contamination of heart tissue fragments.a MHC-GFP à/Thy1+,Isl1-YFP à/Thy1+,a MHC-GFP à/Thy1+/c-kit à,or a MHC-GFP à/Thy1+/c-kit +live cells (as de?ned by the

lack of propidium iodine staining)were isolated using FACS Aria 2(BD Biosci-ences).For conventional isolation of neonatal cardiac ?broblasts,hearts were digested with 0.1%trypsin and plated on plastic dishes (Ieda et al.,2009).For isolation of tail-tip ?broblasts,tails were digested with 0.1%trypsin and plated on plastic dishes.Attached ?broblasts were cultured for 7days and a MHC-GFP à/Thy1+or Mesp1-YFP à/Thy1+cells were sorted and cultured in DMEM/M199medium containing 10%FBS at a density of 104/cm 2.Cells were transduced by retroviruses or lentiviruses after 24hr.

Isolation of Cardiomyocytes

To isolate cardiomyocytes,neonatal a MHC-GFP +ventricles were cut into small pieces and digested with collagenase type II solution (Ieda et al.,2009).A single-cell suspension was obtained by gentle triturating and passing through a 40m m cell strainer.a MHC-GFP +live cells were isolated by FACS Aria 2.To obtain cardiac cells,cells were plated on gelatin-coated plastic dishes and treated with Ara C (Sigma)to inhibit nonmyocyte proliferation.Molecular Cloning and Retroviral/Lentiviral Infection

Retroviruses or inducible lentiviruses containing the cardiac developmental factors were generated as described and as detailed in the Extended Experi-mental Procedures (Kitamura et al.,2003;Takahashi and Yamanaka,2006).The pMXs-DsRed Express retrovirus infection in cardiac ?broblasts resulted in >95%transduction ef?ciency (Hong et al.,2009).

FACS Analyses and Sorting

For GFP expression analyses,cells were harvested from cultured dishes and analyzed on a FACS Calibur (BD Biosciences)with FlowJo software.For a MHC-GFP/cTnT expression,cells were ?xed with 4%PFA for 15min,per-meabilized with Saponin,and stained with anti-cTnT and anti-GFP antibodies,

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followed by secondary antibodies conjugated with Alexa488and647(Katt-man et al.,2006).

For a MHC-GFPà/Thy1+,Isl1-YFPà/Thy1+,and Mesp1-YFPà/Thy1+cell sorting,cells were incubated with PECy7or APC-conjugated anti-Thy1 antibody(eBioscience)and sorted by FACS Aria2(Ieda et al.,2009). For a MHC-GFPà/Thy1+/c-kitàand a MHC-GFPà/Thy1+/c-kit+cell sorting, PECy7-conjugated anti-Thy1and APC-conjugated anti-c-kit antibodies (BD)were used.We used bone marrow cells as a positive control for c-kit staining.

Cell Transplantation

Fibroblasts were harvested1day after retroviral infection.A left thoracotomy was carried out in NOD-SCID mice,and106cultured cells were injected into the left ventricle.After1–2weeks,the hearts were excised for immunohistochemistry.

Histology and Immunocytochemistry

Cells or tissues were?xed,processed and stained with antibodies against numerous proteins in standard fashion as detailed in the Extended Experi-mental Procedures.

Quantitative RT-PCR

Total RNA was isolated from cells,and qRT-PCR was performed on an ABI 7900HT(Applied Biosystems)with TaqMan probes(Applied Biosystems), which are listed in the Extended Experimental Procedures.To quantify endog-enous-speci?c transcripts and both endogenous and transgene common tran-scripts,primers were designed using Vector NTI,and SYBR green technology was used.Primer information is available on request.mRNA levels were normalized by comparison to Gapdh mRNA.

Microarray Analyses

Mouse genome-wide gene expression analyses were performed using Affymetrix Mouse Gene1.0ST Array.a MHC-GFP+cardiomyocytes were collected by FACS.Three-factor transduced GFP+cells and GFPàcells were collected by FACS after2and4weeks of culture.Cardiac?broblasts were also collected after4weeks of culture.RNA was extracted using Pico-Pure RNA Isolation(Arcturus).Microarray analyses were performed in triplicate from independent biologic samples,according to the standard Affymetrix Genechip protocol.Data were analyzed using the Affymetrix Power Tool (APT,version1.8.5).See the Extended Experimental Procedures for additional statistical methods.

Chromatin Immunoprecipitation Assay

Chromatin immunoprecipitations were performed on cardiac?broblasts,tail-tip dermal?broblasts,iCMs,and neonatal cardiac cells.Immunoprecipitations were done using the Imprint Chromatin Immunoprecipitation Kit(Sigma) following the manufacturer instructions.Antibodies against H3K27me3and H3K4me3were from Active motif,and normal rabbit IgG was from Cell Signaling Technology.Primer sequences for qPCR custom TaqMan gene expression assays(Applied Biosystems)are listed in the Extended Experi-mental Procedures.

Bisul?te Genomic Sequencing

Bisul?te treatment was performed using the Epitect Bisul?te Kit(QIAGEN) according to the manufacturer’s recommendations.PCR primers are listed in the Extended Experimental Procedures.Ampli?ed products were cloned into pCR2.1-TOPO(Invitrogen).Ten randomly selected clones were sequenced with the M13forward and M13reverse primers for each gene.

Ca2+Imaging

Ca2+imaging was performed according to the standard protocol.Brie?y,cells were labeled with Rhod-3(Invitrogen)for1hr at room temperature,washed, and incubated for an additional1hr to allow de-esteri?cation of the dye. Rhod-3-labeled cells were analyzed by Axio Observer(Zeiss)with MiCAM02 (SciMedia).Electrophysiology

After4week transduction with GMT,the electrophysiological activities of iCMs were analyzed using extracellular electrode recording with an Axopatch700B ampli?er and the pClamp9.2software(Axon Instruments).iCMs were visually identi?ed by GFP expression and spontaneous contraction.Glass patch pipettes,with typical resistances of2–4M U,were directly attached on single GFP+cells for extracellular recording in Tyrode’s bath solution.For recording intracellular action potentials,single GFP+cells were held atà70mV membrane potential with a stimulation of0.1–0.5nA for5ms to elicit a response after10-week transduction with GMT.

Statistical Analyses

Differences between groups were examined for statistical signi?cance using Student’s t test or ANOVA.p values of<0.05were regarded as signi?cant.

ACCESSION NUMBERS

Microarray data have been submitted and can be accessed by the Gene Expression Omnibus(GEO)accession number GSE22292.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures,?ve ?gures,two tables,and four movies and can be found with this article online at doi:10.1016/j.cell.2010.07.002.

ACKNOWLEDGMENTS

We are grateful to members of the Srivastava lab,to K.Tomoda for critical discussions and comments on the manuscript,to J.Olgin and C.Ding for elec-trophysiology assistance,to Z.Yang and K.Worringer for help with lentivirus experiments,to Y.Huang for cell transplantation experiments,to B.Taylor, G.Howard,and S.Ordway for editorial assistance and manuscript prepara-tion,to C.Barker and L.Ta in the Gladstone Genomics core,to https://www.wendangku.net/doc/4d13014017.html,ler and J.Fish in the Gladstone Histology core,to A.Holloway in the Gladstone Bioin-formatics core,and to S.Elmes in the Laboratory for Cell Analysis in UCSF.We also thank S.Yamanaka for helpful discussions and providing pMXs-DsRed Express plasmid,T.Kitamura for Plat-E cells,J.Robbins for a-MHC promoter plasmid,T.M.Jessell for Isl1-Cre mice,and F.Costantini for R26R-EYFP mice. M.I.and Y.H.are supported by a grant from the Uehara Memorial Foundation. V.V.is supported by grants from the GlaxoSmithKline Cardiovascular Research and Education Foundation and the NIH/NHLBI.P.D.-O.was a post-doctoral scholar of the California Institute for Regenerative Medicine.D.S.and B.G.B are supported by grants from NHLBI/NIH and the California Institute for Regenerative Medicine.The J.David Gladstone Institutes received support from a National Center for Research Resources Grant RR18928-01.D.S.is a member of the Scienti?c Advisory Board of iPierian,Inc.,and RegeneRx.

B.G.B.is a member of the Scienti?c Advisory Board of iPierian Inc.

Received:February18,2010

Revised:May18,2010

Accepted:June25,2010

Published:August5,2010

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